RESUMEN
Orai1 is the pore subunit of Ca(2+) release-activated Ca(2+) (CRAC) channels that stimulate downstream signaling pathways crucial for T cell activation. CRAC channels are an attractive therapeutic target for alleviation of autoimmune diseases. Using high-throughput chemical library screening targeting Orai1, we identified a novel class of small molecules that inhibit CRAC channel activity. One of these molecules, compound 5D, inhibited CRAC channel activity by blocking ion permeation. When included during differentiation, Th17 cells showed higher sensitivity to compound 5D than Th1 and Th2 cells. The selectivity was attributable to high dependence of promoters of retinoic-acid-receptor-related orphan receptors on the Ca(2+)-NFAT pathway. Blocking of CRAC channels drastically decreased recruitment of NFAT and histone modifications within key gene loci involved in Th17 differentiation. The impairment in Th17 differentiation by treatment with CRAC channel blocker was recapitulated in Orai1-deficient T cells, which could be rescued by exogenous expression of retinoic-acid-receptor-related orphan receptors or a constitutive active mutant of NFAT. In vivo administration of CRAC channel blockers effectively reduced the severity of experimental autoimmune encephalomyelitis by suppression of differentiation of inflammatory T cells. These results suggest that CRAC channel blockers can be considered as chemical templates for the development of therapeutic agents to suppress inflammatory responses.
Asunto(s)
Canales de Calcio/metabolismo , Señalización del Calcio , Receptores Nucleares Huérfanos/metabolismo , Células Th17/citología , Células Th17/metabolismo , Animales , Bloqueadores de los Canales de Calcio/química , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/genética , Diferenciación Celular/efectos de los fármacos , Línea Celular , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Humanos , Iones/metabolismo , Ratones , Factores de Transcripción NFATC/metabolismo , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Proteína ORAI1 , Receptores Nucleares Huérfanos/deficiencia , Receptores Nucleares Huérfanos/genética , Regiones Promotoras Genéticas , Unión Proteica , Elementos de Respuesta , Bibliotecas de Moléculas Pequeñas , Células TH1/citología , Células TH1/inmunología , Células TH1/metabolismo , Células Th17/inmunología , Células Th2/citología , Células Th2/inmunología , Células Th2/metabolismoRESUMEN
Orai1 and stromal interaction molecule (STIM)1 are critical components of Ca(2+) release-activated Ca(2+) (CRAC) channels. Orai1 is a pore subunit of CRAC channels, and STIM1 acts as an endoplasmic reticulum (ER) Ca(2+) sensor that detects store depletion. Upon store depletion after T-cell receptor stimulation, STIM1 translocates and coclusters with Orai1 at sites of close apposition of the plasma membrane (PM) and the ER membrane. However, the molecular components of these ER-PM junctions remain poorly understood. Using affinity protein purification, we uncovered junctate as an interacting partner of Orai1-STIM1 complex. Furthermore, we identified a Ca(2+)-binding EF-hand motif in the ER-luminal region of junctate. Mutation of this EF-hand domain of junctate impaired its Ca(2+) binding and resulted in partial activation of CRAC channels and clustering of STIM1 independently of store depletion. In addition to the known mechanisms of STIM1 clustering (i.e., phosphoinositide and Orai1 binding), our study identifies an alternate mechanism to recruit STIM1 into the ER-PM junctions via binding to junctate. We propose that junctate, a Ca(2+)-sensing ER protein, is a structural component of the ER-PM junctions where Orai1 and STIM1 cluster and interact in T cells.
Asunto(s)
Canales de Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de la Membrana/metabolismo , Oxigenasas de Función Mixta/metabolismo , Proteínas Musculares/metabolismo , Proteínas de Neoplasias/metabolismo , Calcio/metabolismo , Canales de Calcio/genética , Proteínas de Unión al Calcio/genética , Membrana Celular/metabolismo , Motivos EF Hand/genética , Retículo Endoplásmico/metabolismo , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Células Jurkat , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas de la Membrana/genética , Microscopía Fluorescente , Oxigenasas de Función Mixta/genética , Proteínas Musculares/genética , Mutación , Proteínas de Neoplasias/genética , Proteína ORAI1 , Unión Proteica , Transporte de Proteínas , Molécula de Interacción Estromal 1RESUMEN
Sustained activation of the Ca2+-release-activated Ca2+ (CRAC) channel is pivotal for effector T cell responses. The mechanisms underlying this sustainability remain poorly understood. We find that plasma membrane localization of ORAI1, the pore subunit of CRAC channels, is limited in effector T cells, with a significant fraction trapped in intracellular vesicles. From a targeted screen, we identify an essential component of ORAI1+ vesicles, naked cuticle homolog 2 (NKD2). Mechanistically, NKD2, an adaptor molecule activated by signaling pathways downstream of T cell receptors, orchestrates trafficking and insertion of ORAI1+ vesicles to the plasma membrane. Together, our findings suggest that T cell receptor (TCR)-stimulation-dependent insertion of ORAI1 into the plasma membrane is essential for sustained Ca2+ signaling and cytokine production in T cells.